Found a new "Ultimate" guide for molecular cloning construct design. It seems useful to most researchers.
This Molecular Cloning Guides covers:
Subcloning is a technique used to move a particular gene of interest from a parent vector to a destination vector in order to further study its functionality.
Use PCR reactions to amplify a insert DNA fragment, followed by sub-cloning (restriction endonuclease digestion of insert and vector DNA and fragment ligation).
Use DNA oligos and PCR reactions to synthesize a gene without source DNA as template followed by Subcloning (restriction endonuclease digestion and fragment ligation).
TA TOPO Cloning:
TA Cloning is a subcloning technique that doesn't use restriction enzymes and is easier and quicker than traditional subcloning.
Directional TOPO Cloning:
Use PCR reaction to amplify a DNA fragment. The resulting PCR products have four additional bases (CACC) at the 5´ ends that are from the specially designed forward PCR primer. With a special ligation kit, this fragment is directly ligated into a linearized vector DNA (D-TOPO Vector, which contains GTGG overhangs at the 5’ end) without pre-digestion with restriction endonucleases. The fragment can only be inserted in forward orientation.
This is a cloning method based on the site specific recombination of lambda bacteriophage.
Found a new Ultimate guide for molecular cloning construct design
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